EP0430719A2 - Composés et réactifs pour la détermination de réticulocytes - Google Patents

Composés et réactifs pour la détermination de réticulocytes Download PDF

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Publication number
EP0430719A2
EP0430719A2 EP90313101A EP90313101A EP0430719A2 EP 0430719 A2 EP0430719 A2 EP 0430719A2 EP 90313101 A EP90313101 A EP 90313101A EP 90313101 A EP90313101 A EP 90313101A EP 0430719 A2 EP0430719 A2 EP 0430719A2
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EP
European Patent Office
Prior art keywords
derivative
acridine orange
reticulocytes
dye
dye composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP90313101A
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German (de)
English (en)
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EP0430719B1 (fr
EP0430719A3 (en
Inventor
Sophie S. Fan
Gena Fischer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bayer Corp
Original Assignee
Bayer Corp
Technicon Instruments Corp
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Publication date
Application filed by Bayer Corp, Technicon Instruments Corp filed Critical Bayer Corp
Publication of EP0430719A2 publication Critical patent/EP0430719A2/fr
Publication of EP0430719A3 publication Critical patent/EP0430719A3/en
Application granted granted Critical
Publication of EP0430719B1 publication Critical patent/EP0430719B1/fr
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B15/00—Acridine dyes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/145555—Hetero-N

Definitions

  • the present invention relates to the use of certain quaternized derivatives of acridine orange for quantitatively determining reticulocyte levels in whole blood, to dye compositions useful in such determinations, and to certain quaternized acridine orange derivatives per se.
  • the blood comprises an aqueous fluid part (the plasma) in which are suspended corpuscles of various kinds: the red blood cells (erythrocytes), the white blood cells (leukocytes) and the blood platelets.
  • Plasma has a composition comprising roughly 90% water, 9% protein, 0.9% salts and traces of other materials such as sugar, urea, uric acid and the like.
  • the cells or corpuscles of the peripheral blood i.e. the blood outside the bone marrow
  • the cells or corpuscles of the peripheral blood are divided into two main groups: the red blood cells (erythrocytes), whose primary object is to transport oxygen, and the white blood cells (leukocytes), whose primary functions relate to the immune system and the destruction of materials foreign to the body.
  • the blood also contains the so-called blood platelets which are important in hemostatis.
  • reticulocytes The final stages of erythrocyte maturation occur after their release from the bone marrow while these cells are circulating in the peripheral blood. These young red cells, or "reticulocytes", have lost their nucleus and thus their ability to divide or to synthesize RNA. Although these functions have ceased, reticulocytes are still metabolically active and are capable of synthesizing protein, taking up iron for the synthesis of heme, and carrying out the necessary metabolic reactions required to maintain an energy rich state. These cells are usually distinguished from mature erythrocytes through the presence of the reticulum which gives them their name. This reticulum may be dyed by such agents as brilliant cresyl blue, nile blue sulfate or new methylene blue, after which quantitation of reticulocytes may be performed by way of manual observation under a microscope.
  • reticulocytes normally comprise about 0.5 to 2 percent of the total red blood cell population, this percentage can change dramatically under abnormal conditions.
  • reticulocyte counts have been used for many years as a diagnostic aid in studying blood dyscrasias and as an index of red cell regeneration following haemorrhage, as well as for monitoring early toxicity in chemotherapy of certain malignant diseases.
  • the Adams and Kamentsky patent describes the use of a vital dye composition for differential blood analysis of living white cells which consists essentially of acridine orange having a concentration between 10 ⁇ 7 and 10 ⁇ 5 grams per ml, the acridine orange solution having a pH factor and an osmolality within the normal physiological ranges for human blood plasma. While the patent teaches that this composition is useful for identifying the various types of white blood cells and for distinguishing them from other bodies in the blood, there is no teaching that this composition has any use in the enumeration of reticulocytes.
  • the Adams patent represents a modification of the teaching of Adams and Kamentsky in that the white blood cells are treated under conditions in which the cells are "shocked" by exposure to a non-physiological medium during staining. That is, the staining composition used in the Adams patent is made hypotonic, the osmolality thereof being generally below that normally found in human blood.
  • the teaching of the Adams patent is that this hypotonic condition produces a differential rate of uptake of acridine orange dye by the various types of white blood cells, thus allowing them to be more clearly distinguished from one another than in previous techniques.
  • the Adams patent does purport to disclose a method for the detection of reticulocytes, the method disclosed therein has been criticized as being practically useless for the quantitation of reticulocytes in U.S. Patent No. 4,336,029 to Natale.
  • the invention of the Natale patent depends upon removing the kinetic factors and increasing the degree of dye uptake so that the reticulocytes will absorb a maximum amount of acridine orange dye.
  • the reticulocytes absorb only small amounts of dye and therefore yield only low levels of fluorescence in any fluorescence detecting method. These low levels of fluorescence could generally not be well detected over the background fluorescence and consequently only a portion of reticulocytes in the sample could be detected.
  • the dye composition of the Natale patent consists essentially of an aqueous solution of the metachromatic fluorochrome dye acridine orange, a chelating agent (citrate), an amino-group reacting reagent, and (if needed) a buffer to maintain the final pH of this solution at approximately 7.4
  • the osmolality of the solution is maintained at approximately 0.26 osmolality units, the normal physiological level, either by the chelating agent or by addition of sodium chloride as required.
  • the main purpose of the Natale teaching is to identify both reticulocytes and platelets simultaneously.
  • the Natale reagent contains citrate ions to maximize platelet staining.
  • the Natale reagent contains very high concentrations of the acridine orange dye (10 ⁇ 2 gram per liter) which has been found to stain the fluid conduits within the instrument, including the flow cells, resulting in false positive readings for white cells. This staining also creates carryover problems requiring extensive washing of the system which adds time consuming steps to the assay protocol.
  • acridine orange specifically teach against having maximum acridine orange uptake, since such maximum uptake destroys the discrimination among the various subclasses of white blood cells which is the main object of these prior art methods.
  • a dye composition for the quantitation of reticulocytes in a whole blood sample which composition comprises an aqueous solution of a quaternized derivative of acridine orange of the formula: wherein X is a benzyl group of formula wherein R1 is hydrogen or fluorine, and R2 is fluorine, trifluoromethyl or hydrogen, or X is a hydroxyethylene group; and ⁇ is bromide or iodide.
  • the invention also includes a method of quantitating reticulocytes in a whole blood sample by flow cytometry, which comprises the steps of:
  • acridine orange derivatives used in the present invention are novel. These compounds are of formula I as defined above but R1 and R2 are not both hydrogen.
  • X can be a benzyl group of the formula wherein R1 is hydrogen or fluorine, and R2 is fluorine, trifluoromethyl (CF3) or hydrogen; to form the compound of the general formula (II): where ⁇ is preferably bromide.
  • X may be a hydroxyethylene group [(CH2)2OH] to form the compound of the general formula (III) where ⁇ is preferably iodide.
  • the dye compositions (sometimes called reagent compositions) of the invention preferably comprise a buffer to maintain the pH at about neutral (7.0).
  • a preferred buffer comprises paraformaldehyde and potassium oxalate, but others can be used.
  • from 3 to 9 ⁇ g/ml of the acridine orange derivative is present in a buffer solution comprising 1.25 g/l paraformaldehyde and 9 g/l potassium oxalate.
  • flow cytometry is used to measure the reticulocytes in a whole blood specimen.
  • the fundamental concept of flow cytometry is essentially that of passing cells, one at a time, through a specific sensing region.
  • the sensing zone which consists of a focussed laser light source and a detection system for the measurement of scattered and fluorescent light.
  • the technique will be familiar to those skilled in the art.
  • the blood sample is mixed with a composition of the invention, and the mixture (which is generally a suspension) is allowed to stand at room temperature for a short time, for example, three minutes or less, so that the acridine orange dye is maximally taken up by the reticulocytes. The red or orange fluorescence is then measured.
  • a composition of the invention which is generally a suspension
  • Example 1 The procedure of Example 1 was followed, with different alkylation agents used at the same molar concentration. Fluorinated benzyl groups were substituted at either the 2- or 4- positions in the benzene ring.
  • the alkylation agents included the following:
  • Example 3 Synthesis of 3,6-bis(dimethylamino)-10-ethanol acridinium derivative.
  • Example 5 pH titration of the Derivative (II) Family and Derivative (III).
  • Example 6 Fluorescence binding of the Derivative (II) Family and Derivative (III) to RNA (Saturated Solution).
  • RNA solution Calbiochem Cat. No. 557112
  • concentrations of 100 mg/ml in PBS buffer were added and mixed with 3 ml of an acridine dye derivative solution of the invention (1 ⁇ g/ml dye concentration).
  • the fluorescence spectrum of each was recorded until the dye solution was saturated with RNA. This was found to occur when 40 ⁇ l or greater volume of RNA solution had been added to the dye solution.
  • Table 2 shows the metachromatic fluorescence shift and fluorescence enhancement factor for the dye compositions of the invention as compared to acridine orange.
  • the data demonstrate a greater shift for the compounds of the invention than for acridine orange, and the same or a greater increase in fluorescence signal when bound to RNA.
  • the ratio of the fluorescence signal of the bound dye to that of the free dye in solution is referred to as the Fluorescence Enhancement Factor.
  • Example 7 Microscopic examination of reticulocyte staining with the Derivative (II) Family and Derivative (III)
  • Protocol for sample preparation About 20 ⁇ l of an acridine derivative dye solution (stock solution of 0.3 mg/ml to 0.9 mg/ml of the compound in 100% methanol) were added to 2 ml of a buffer solution including 0.9% potassium oxalate and 1.25 mg/ml paraformaldehyde and mixed thoroughly. To this solution, 20 ⁇ l of either normal or patient blood sample were added, vortexed and held at room temperature for three minutes. The samples were then ready for reticulocyte detection. For enriched reticulocyte samples, 10 ⁇ l of blood was suspended in the 2 ml reagent. For acridine orange staining solution (used for comparison), the stock solution contained 3 mg/ml acridine orange.
  • NMB New Methylene Blue
  • NCLS National Committee for Clinical Laboratory Standards
  • reticulocytes which showed red precipitates of RNA were clearly observed for those blood samples stained with Derivative (IIa), (IIb), (IIc), (IId) and (III).
  • acridine orange-10-2-nitro-benzyl and acridine orange-10-2-chloro-benzyl did not stain the reticulocytes specifically.
  • acridine orange was used as a reference to show that not all substituents at the benzyl hydrogen can stain reticulocytes. It was noticed that the amount of acridine orange needed for good reticulocyte staining was about 10-14 times higher than the amount of Derivative (II) or (III) needed.
  • Example 8 Stability of the reagent performance within days using Derivative (IIa).
  • a reagent containing Derivative (IIa) was compared with one containing acridine orange.
  • One blood sample (normal) was stained with the dye solutions separately.
  • the protocol for the sample preparation was as described for Example 7.
  • a Spectrum III flow cytometer was used for this evaluation. After blood was added to the dye solutions, the two different mixtures were left at room temperature and exposed to ambient light. Reticulocyte counting of the two mixtures was conducted at different time intervals for up to eight hours. At each interval, the measurement was run in duplicate. Table 4 below summarizes the results.
  • Derivative (IIa) gives more reproducible counts in eight hours.
  • the 10.7% CV for Derivative (IIa) is to be compared with the 16% CV for acridine orange.
  • Example 9 Stability of the reagents (shelf life).
  • the stabilities of two reagents were studied and compared.
  • a 3 ⁇ g/ml concentration of Derivative (IIa) and 30 ⁇ g/ml of acridine orange each in the buffer solution of Example 7 were left at room temperature unprotected from ambient light for a week.
  • the absorption and fluorescent spectra of the dye solutions were recorded each day.
  • Derivative (IIa) has the least drop in both fluorescence and absorption intensities after six days. About 30% decrease in both fluorescence and absorption was observed for the benzyl derivative and 70% decrease for acridine orange. Thus, Derivative (IIa) is more stable and gives more reproducible reticulocyte counts than acridine orange.
  • Example 10 Correlation study with NCCL's manual reference method.
  • Fig. 7 shows a correlation coefficient of 0.99 and a slope of 1.06 and intercept of 0.13% based on a nine patient sample.
  • Example 11 Correlation with a commercial automated cytometric method.
  • Example 8 The protocols of Example 8 were followed for all sample measurements using the Spectrum III system.
  • Fig. 8a shows the result: correlation coefficient 0.88, slope 1.45 and intercept-1.4%. Since the B-D FACScan method is a commercialized cytometric method, these results indicate comparability of the claimed reagent to the recognized cytometric counting method.
  • Figure 9 demonstrates the high degree of discrimination between reticulocyte and erythrocyte populations when cells were stained with Derivative (III), and measured by the Spectrum III instrument.
  • the sample preparation protocol is the same as discussed above. Distinct cell populations were clearly observed based on their particular scatter and fluorescence signals.
  • the erythrocyte population falls within Region A between the vertical axis and vertical line X. These cells show high scatter signals and low cell fluorescence signals.
  • the reticulocyte population falls within Region B (to the right of X). These cells are distinguishable from the erythrocytes due to the high fluorescence signals from their Derivative (III) stained RNA.
  • the platelet population lies within Region C below line Y.
  • the reticulocyte count of a patient sample was measured to be 2.2%.
  • the same blood sample was also analyzed by the NCCLS method. The result was a reticulocyte count of 2.6%.

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EP90313101A 1989-12-01 1990-12-03 Composés et réactifs pour la détermination de réticulocytes Expired - Lifetime EP0430719B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US444255 1989-12-01
US07/444,255 US5075556A (en) 1989-12-01 1989-12-01 Acridine orange derivatives and their use in the quantitation of reticulocytes in whole blood

Publications (3)

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EP0430719A2 true EP0430719A2 (fr) 1991-06-05
EP0430719A3 EP0430719A3 (en) 1992-02-05
EP0430719B1 EP0430719B1 (fr) 1995-10-04

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US (1) US5075556A (fr)
EP (1) EP0430719B1 (fr)
JP (1) JPH0822967B2 (fr)
AU (1) AU626508B2 (fr)
CA (1) CA2024166C (fr)
DE (1) DE69022819T2 (fr)
DK (1) DK285390A (fr)
ES (1) ES2080127T3 (fr)
IL (1) IL95485A (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0545313A1 (fr) * 1991-12-05 1993-06-09 Bayer Corporation Compositions d'agents réactifs et leur utilisation pour créer des cellules rouges du sang de forme sphérique
EP0545315A1 (fr) * 1991-12-05 1993-06-09 Bayer Corporation Compositions réactives et leur utilisation dans l'identification et caractérisation de réticulocytes du sang entier
EP0545314A1 (fr) * 1991-12-05 1993-06-09 Bayer Corporation Compositions réactives et leur utilisation dans l'identification et caractérisation de réticulocytes du sang entier
WO1997019356A1 (fr) * 1995-11-20 1997-05-29 Abbott Laboratories Systeme de reactif et procede de differentiation et d'identification de reticulocytes
EP0856735A1 (fr) * 1997-01-31 1998-08-05 Abx Réactif de coloration pour la détermination de cellules sanguines
EP1376135A3 (fr) * 2002-05-20 2004-05-19 Bayer Corporation Procédé automatisé et réagent permettant de mesurer des échantillons de liquides biologiques, comme le liquide céphalorachidien
RU2418293C1 (ru) * 2010-01-11 2011-05-10 Государственное образовательное учреждение высшего профессионального образования "Томский государственный университет" (ТГУ) Способ обнаружения йодидов
RU2444724C1 (ru) * 2010-10-21 2012-03-10 Государственное образовательное учреждение высшего профессионального образования "Томский государственный университет" (ТГУ) Способ разделения минералов меди и серебра из зон окисления сульфидных полиметаллических месторождений
CN101910839B (zh) * 2008-02-18 2012-10-17 希森美康株式会社 血小板测定用试剂、血小板测定用试剂盒及血小板测定方法

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JP3070968B2 (ja) * 1991-05-14 2000-07-31 シスメックス株式会社 尿中の細胞分析用試薬及び方法
JP3024301B2 (ja) * 1991-09-18 2000-03-21 栗田工業株式会社 アゾール類の濃度管理方法
US5334509A (en) * 1992-10-22 1994-08-02 Riordan Neil H Method for detecting intestinal pathogen dientamoeba fragilis
US5563070A (en) * 1993-05-28 1996-10-08 Omron Corporation Method of counting reticulocytes
EP0912766B2 (fr) * 1996-06-04 2011-12-14 University of Utah Research Foundation Controle de l'hybridation pendant la pcr
US6114173A (en) * 1997-04-03 2000-09-05 Bayer Corporation Fully automated method and reagent composition therefor for rapid identification and characterization of reticulocytes erythrocytes and platelets in whole blood
US6060322A (en) * 1998-10-20 2000-05-09 Coulter International Corp. Method for identification of reticulated cells
EP1644330B1 (fr) * 2003-06-27 2011-08-17 The University Of Maryland Composes heterocycliques contenant de l'azote quaternaire, servant a detecter des monosaccharides aqueux dans des fluides physiologiques
CN101349644B (zh) 2007-07-20 2012-06-27 深圳迈瑞生物医疗电子股份有限公司 一种白细胞分类试剂和其使用方法
US8102161B2 (en) * 2007-09-25 2012-01-24 Tdk Corporation Stable output in a switching power supply by smoothing the output of the secondary coil
CN101475754A (zh) 2008-01-04 2009-07-08 深圳迈瑞生物医疗电子股份有限公司 不对称菁类荧光染料,组合物及在生物样品染色中的用途
CN101602762B (zh) 2008-06-10 2013-10-16 深圳迈瑞生物医疗电子股份有限公司 不对称菁类化合物、其制备方法及应用
CN101726579B (zh) 2008-10-17 2014-06-18 深圳迈瑞生物医疗电子股份有限公司 血液检测试剂和方法
CN101750274B (zh) 2008-12-17 2014-06-25 深圳迈瑞生物医疗电子股份有限公司 白细胞分类计数试剂、试剂盒以及白细胞分类计数的方法
CN101988082B (zh) 2009-07-31 2015-04-08 深圳迈瑞生物医疗电子股份有限公司 白细胞分类计数试剂、试剂盒及其制备方法和白细胞分类计数的方法
CN112639467B (zh) * 2018-12-25 2024-04-09 深圳迈瑞生物医疗电子股份有限公司 血小板模拟粒子及其制备方法以及含该模拟粒子的质控物或校准物

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US3684377A (en) * 1970-07-13 1972-08-15 Bio Physics Systems Inc Method for analysis of blood by optical analysis of living cells
US3883247A (en) * 1973-10-30 1975-05-13 Bio Physics Systems Inc Method for fluorescence analysis of white blood cells
US4336029A (en) * 1980-08-15 1982-06-22 Ortho Diagnostic Systems Inc. Method and reagents for quantitative determination of reticulocytes and platelets in whole blood

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0545313A1 (fr) * 1991-12-05 1993-06-09 Bayer Corporation Compositions d'agents réactifs et leur utilisation pour créer des cellules rouges du sang de forme sphérique
EP0545315A1 (fr) * 1991-12-05 1993-06-09 Bayer Corporation Compositions réactives et leur utilisation dans l'identification et caractérisation de réticulocytes du sang entier
EP0545314A1 (fr) * 1991-12-05 1993-06-09 Bayer Corporation Compositions réactives et leur utilisation dans l'identification et caractérisation de réticulocytes du sang entier
AU661728B2 (en) * 1991-12-05 1995-08-03 Bayer Corporation Reagent compositions and their use in sphering cells
WO1997019356A1 (fr) * 1995-11-20 1997-05-29 Abbott Laboratories Systeme de reactif et procede de differentiation et d'identification de reticulocytes
EP0856735A1 (fr) * 1997-01-31 1998-08-05 Abx Réactif de coloration pour la détermination de cellules sanguines
FR2759166A1 (fr) * 1997-01-31 1998-08-07 Abx Sa Reactif de coloration pour la determination de cellules sanguines
US5994138A (en) * 1997-01-31 1999-11-30 Abx Staining reagent for the determination of blood cells
EP1376135A3 (fr) * 2002-05-20 2004-05-19 Bayer Corporation Procédé automatisé et réagent permettant de mesurer des échantillons de liquides biologiques, comme le liquide céphalorachidien
CN101910839B (zh) * 2008-02-18 2012-10-17 希森美康株式会社 血小板测定用试剂、血小板测定用试剂盒及血小板测定方法
RU2418293C1 (ru) * 2010-01-11 2011-05-10 Государственное образовательное учреждение высшего профессионального образования "Томский государственный университет" (ТГУ) Способ обнаружения йодидов
RU2444724C1 (ru) * 2010-10-21 2012-03-10 Государственное образовательное учреждение высшего профессионального образования "Томский государственный университет" (ТГУ) Способ разделения минералов меди и серебра из зон окисления сульфидных полиметаллических месторождений

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Publication number Publication date
US5075556A (en) 1991-12-24
CA2024166C (fr) 1995-08-01
DE69022819D1 (de) 1995-11-09
AU6260890A (en) 1991-06-06
DK285390A (da) 1991-06-02
DK285390D0 (da) 1990-11-30
JPH0822967B2 (ja) 1996-03-06
AU626508B2 (en) 1992-07-30
IL95485A0 (en) 1991-06-30
CA2024166A1 (fr) 1991-06-02
DE69022819T2 (de) 1996-03-14
EP0430719B1 (fr) 1995-10-04
ES2080127T3 (es) 1996-02-01
JPH03182562A (ja) 1991-08-08
EP0430719A3 (en) 1992-02-05
IL95485A (en) 1994-04-12

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